The attenuation effect of potassium 2‐(1‐hydroxypentyl)‐benzoate in a mouse model of diabetes‐associated cognitive decline: The protein expression in the brain

Abstract Aims dl‐PHPB (potassium 2‐(1‐hydroxypentyl)‐benzoate) has been shown to have neuroprotective effects against acute cerebral ischemia, vascular dementia, and Alzheimer's disease. The aim of this study was to investigate the effects of dl‐PHPB on memory deficits and preliminarily explore the underlying molecular mechanism. Methods Blood glucose and behavioral performance were evaluated in the KK‐Ay diabetic mouse model before and after dl‐PHPB administration. Two‐dimensional difference gel electrophoresis (2D‐DIGE)‐based proteomics was used to identify differentially expressed proteins in brain tissue. Western blotting was used to study the molecular mechanism of the related signaling pathways. Results Three‐month‐old KK‐Ay mice were given 150 mg/kg dl‐PHPB by oral gavage for 2 months, which produced no effect on the level of serum glucose. In the Morris water maze test, KK‐Ay mice treated with dl‐PHPB showed significant improvements in spatial learning and memory deficits compared with vehicle‐treated KK‐Ay mice. Additionally, we performed 2D‐DIGE to compare brain proteomes of 5‐month KK‐Ay mice treated with and without dl‐PHPB. We found 14 altered proteins in the cortex and 11 in the hippocampus; two of the 25 altered proteins and another four proteins that were identified in a previous study on KK‐Ay mice were then validated by western blot to further confirm whether dl‐PHPB can reverse the expression levels of these proteins. The phosphoinositide 3‐kinase/protein kinase B/glycogen synthase kinase‐3β (PI3K/Akt/GSK‐3β) signaling pathway was also changed in KK‐Ay mice and dl‐PHPB treatment could reverse it. Conclusions These results indicate that dl‐PHPB may play a potential role in diabetes‐associated cognitive impairment through PI3K/Akt/GSK‐3β signaling pathway and the differentially expressed proteins may become putative therapeutic targets.


| INTRODUC TI ON
Type 2 diabetes mellitus (T2DM) is a metabolic disorder and accounts for more than 90% of diabetes patients. As T2DM has an impact on cardiovascular, peripheral nervous systems, and central nervous system (CNS), and cognitive changes mainly affect learning and memory, mental speed and flexibility, 1,2 so diabetic encephalopathy (DE) is put forward. Because of the global prevalence of T2DM, DE has gained much public attention and has become a major public health concern worldwide. It is a series of neuropathological changes caused by diabetes and the common symptoms are paraesthesia, numbness, and impaired cognition. 3 AD and T2DM are two age-related diseases that are increasing worldwide. Alzheimer's disease (AD) is a most common neurodegenerative disorder. The accumulation of brain amyloidβ (Aβ) and hyperphosphorylated tau is implicated in the pathogenesis of AD.
Numerous studies have demonstrated that patients with diabetes have an increased risk of developing AD compared with healthy individuals. 1,4 However, the underlying biological mechanisms that link T2DM and AD are not fully understood. The aggregation of Aβ, disruption of the insulin signaling pathway, increased oxidative stress, dysregulated glucose metabolism, formation of advanced glycation end products (AGEs), and activation of inflammatory pathways all occur in both diseases. [5][6][7][8] Insulin resistance can affect energy metabolism, cell growth and differentiation, cellular repair mechanisms, and glucose utilization. 7 It is a core feature of T2DM and seems to be the main common impairment of the two diseases. In 2009, dl-PHPB was approved to undergo phase I, II, and III clinical trials by the State Food and Drug Administration as a new drug candidate for ischemic stroke. 9,10 Our preliminary studies showed that dl-PHPB could improve cognitive deficits induced by hypoperfusion and also induced by icv infused with Aβ through preventing neuropathological alterations, inhibiting oxidative damage and inflammatory reactions. 10 These results indicated that dl-PHPB had the therapeutic potential for the treatment of vascular dementia (VaD) and AD.
Recently, a lot of evidence has shown that GSK-3β may be the potential link between diabetes mellitus (DM) and AD. 11 In DM, GSK-3β is crucial for PI3K/Akt/insulin signaling pathway and is involved in the glucose metabolism in the brain. 12 PI3K/Akt/GSK-3β signaling pathway also plays an important role in hyperphosphorylation of the microtubule-associated protein tau (tau) which is one of the important pathological features in AD. 13 Our previous study showed that dl-PHPB could upregulate PI3K/Akt signaling pathway and downregulated the expression of phosphorylated GSK-3β in APP and presenilin 1 (PS1) double-transgenic (APP/PS1) AD mice and further reduced hyperphosphorylation of tau protein. 14 All existing evidence shows that dl-PHPB has the potential to improve memory deficits in VaD and AD. Therefore, we speculate that it may also have a therapeutic effect on DE. The technique of 2D-DIGE combined the traditional two-dimensional electrophoresis technology with its characteristic of multiple fluorescence analysis technology. In this study, we investigated the pharmacological activity of dl-PHPB on cognitive impairment in a T2DM animal model, KK-A y mice, and examined alterations in the cortical and hippocampal protein profile in mice with DE and in dl-PHPB-treated mice using 2D-DIGE-based proteomics. Moreover, we tested the PI3K/Akt/ GSK-3β pathway to investigate the possible underlying molecular mechanism.

| Animals and drug administration
dl-PHPB (purity > 98%) was offered by the Department of Medical Synthetic Chemistry, Institute of Material Medic (Beijing, China), and dissolved in distilled water. Its chemical structure is shown in Figure 1.
Adult male KK-A y mice and age-matched C57 BL/6J mice were used in this study. Mice were randomly divided into three groups: C57 group (oral gavage distilled water), KK-A y group (oral gavage distilled water), and KK-A y + dl-PHPB group (oral gavage dl-PHPB 150 mg/kg). Distilled water and dl-PHPB were administered to mice every day for a total of 2 months. Mice were kept in a temperaturecontrolled room (23-25°C) under a 12-h/12-h light/dark cycle and free access to food and water. The C57 group was fed a standard diet and the other two groups were given a high-fat diet (40% energy from fat) until the mice were sacrificed. Tail random blood glucose was measured when we obtained these mice and monitored every 2 weeks. All animal experiments were performed under the institutional guidelines and regulations of the Experimental Animal Center of the Chinese Academy of Medical Science, Beijing, China.

| Morris water maze test
The Morris water maze test was proceeded to evaluate the mouse spatial learning and memory performance at the age of 21 weeks after dl-PHPB administration. The device consists of a circular stainless steel water tank (120 cm in diameter and 50 cm in height).
The tank was filled with water to a depth of 30 cm and the water's temperature was kept at 24 ± 1°C. A transparent acrylic platform F I G U R E 1 Chemical structure of dl-PHPB (10 cm in diameter, hidden below the water surface about 1.5 cm) was placed at the center of the southwest quadrant during training.
Obvious clues were on the walls of the room for the mice to orientate. The swimming paths were recorded by a video camera and data were automatically collected into a computer with an image analyzer immediately. The orientation navigation test lasted for 5 days with the location of the platform fixed, but the starting positions were changed every day. On each day, mice were trained with two consecutive trials and the intertrial interval was 30 s. The mice were permitted a maximum of 120 s to find the hidden platform and were allowed to rest on the platform for 30 s. If the mouse failed to climb on the platform itself within 120 s, it would be guided to the platform by the experimenter gently. During the test, the swimming pathway and escape latency of mice were recorded. On the 6th day, to assess long-term memory consolidation, the platform was removed and the mice were placed in the pool from the opposite quadrant where the platform was located before. The mice were allowed to search for the platform for 120 s. The time that an animal crossed the position of the hidden platform at the first time, the time spent in the target quadrant and crossing times in the platform quadrant were recorded to measure the spatial memory maintenance.

| Sample collection and preparation
After Morris water maze test, mice were anesthetized with 4% (w/w)

| Proteins labeling with CyDyes and twodimensional electrophoresis
A quantity of 50 μg of protein sample from the cortex or hippocampus of each mouse (n = 6) was added to a tube and labeled with 400 pmol Cy3 or Cy5 minimal dye. To avoid possible dye bias, dyes were designed to swap within the two groups. A pooled internal standard consisting of equal amounts of each sample was created and labeled with Cy2 minimal dye. This internal standard would need to be sufficient to include on every gel. Centrifuged briefly in a microcentrifuge to make the solution collected at the bottom of the tube and left for 30 min on ice in the dark. Added 1 μl of 10 mM lysine to the sample and left for 10 min on ice in the dark to stop the reaction.
The main difference between 2D-DIGE and standard 2-DE (twodimensional electrophoresis) techniques is that up to three different protein samples can be run on a single gel. We mixed together three differently labeled protein samples (an internal standard labeled with Cy2, a diabetic sample and a control sample labeled with Cy3 or Cy5 respectively) and added an equal volume of 2× sample buffer con- The total volume of labeled protein was made up to 350 μl with the rehydration buffer consisting of 4% (w/v) CHAPS, 8 M urea, 1% (v/v) IPG buffer, 13mM DTT, and 0.002% (w/v) Bromophenol blue.
Isoelectric focusing was carried out using the Ettan IPGphor II IEF system (GE Healthcare) and 18 cm pH 3-10 nonlinear immobiline drystrips were used. IEF was run at 20°C overnight and finally achieving 60,000V h at a maximum current of 50 μA per strip.

Second dimension separation was performed on an Ettan DALTsix
Electrophoresis System (GE Healthcare) at 9°C using 2 W per gel for 1 h and then 15W per gel until the bromophenol blue dye front reached the bottom of the gel.
Gel image analysis was performed by using DeCyder 2D Software (GE Healthcare) which enables detection, quantification, matching and analysis of 2D-DIGE system gels. The steps consist of the following processes: spot detection, background subtraction, in-gel normalization, gel artifact removal, gel to gel matching, statistical analysis. Protein spots of interest with significant difference (average ratio ≥ 1.5 and p < 0.5) were filtered and a pick list was created. Then, we excised these proteins manually on a gel stained with Coomassie blue which separated unlabeled pooled protein samples for further mass spectrometry analysis. Appraisal of each protein should have at least two specific peptides to support.

| Western blot analysis
The expression levels of differentially expressed proteins were detected by western blot according to the protocol as described previously. 60 μg protein per lane was run on a polyacrylamide gel, transferred on to a PVDF membrane, blocked with 5% milk solution (nonfat dry milk in TBST) for 2 h and subsequently incubated with primary antibodies diluted in blocking solution overnight. The following primary antibodies were used in this study: rabbit mono-

| Statistical analysis
Statistical tests were performed by SPSS version 16.0 software. All the data are expressed as mean ± SEM. Data normality was verified by Shapiro-Wilk test. The results of behavioral tests were analyzed by two-way repeated measures ANOVA followed by the Bonferroni F I G U R E 2 Body weight (BW), serum glucose (Glu) of C57 mice, KK-A y mice, and KK-A y + dl-PHPB during drug application for 2 months. n = 15 for the C57 group, n = 20 for the KK-A y group, and n = 19 for KK-A y + dl-PHPB group. ## p < 0.01 vs C57 group F I G U R E 3 dl-PHPB attenuates cognitive impairment of KK-A y mice in the Morris water maze test. (A) Time course of escape latency from day 1 to day 5. (B) The time that an animal crossed the position of the hidden platform at the first time on day 6. (C) Crossing times in the platform quadrant on day 6. n = 15 for C57 group and n = 20 for KK-A y group, n = 19 for KK-A y + dl-PHPB group. ## p < 0.01 and # p < 0.05 vs C57 group, * p < 0.05 vs KK-A y group post hoc test. One-way ANOVA analysis followed by the Tukey post hoc test was performed to analyze statistical difference for multiple group comparisons. p-Value < 0.05 was considered to be statistically significant.

| Serum index analysis
The body weight (BW) and serum glucose (Glu) levels were significantly higher (p < 0.01) in KK-A y mice than in C57 mice at 5 months of age ( Figure 2). However, administration of dl-PHPB to KK-A y mice had no effect on these indexes compared with KK-A y mice.

| Cognitive performance in the Morris water maze test
Our previous studies revealed that KK-A y mice showed cognitive deficits in the Morris water maze test and synaptic plasticity beginning at 3 months of age. 16 To test the effect of dl-PHPB on cognitive impairment, we evaluated the orientation navigation task using the Morris water maze. The spatial learning ability was measured by escape latency (the time required to reach the hidden platform). Figure 3A shows that during the 5 training days, the escape latency of the C57 group was shorter than that of the KK-A y group, and repeated-measures ANOVA showed a significant effect of day on escape latency (p < 0.01), indicating spatial memory impairment. In contrast, KK-A y mice treated with dl-PHPB showed reduced escape latency, indicating that dl-PHPB can improve the spatial learning and memory impairment of KK-A y mice. In the probe trial, dl-PHPB also increased the crossing times of platform location and decrease the first crossing time of platform location, as compared with KK-A y mice, but not significantly ( Figure 3B,C). These results show that dl-PHPB can attenuate the spatial learning and memory deficits of KK-A y mice.

| Fluorescence-based DIGE and protein identification in cortical tissue from KK-A y mice and KK-A y +dl-PHPB mice
For each gel, more than 5000 spots were detected using DeCyder 2D software. Differentially expressed protein spots are labeled with black circles and corresponding numbers in Figure 4A.  Figure 5B and Figure 5C Table 1.   Table 2. We also examined the expression levels of the six proteins (GNAO1, HINT1, HAGH, EB1, DRP2, and LAMTOR2) in the hippocampus. Representative immunoblot images ( Figure 8A) and bar graphs ( Figure 8B) of the hippocampal brain homogenates of C57 mice, KK-A y mice, and KK-A y +dl-PHPB mice are displayed.
Compared with the C57 group, the expression of the six proteins in the KK-A y group tended to be downregulated, but only GNAO1 and HAGH were significantly downregulated. In the KK-A y + dl-PHPB group, only the expression levels of EB1 and HAGH were significantly upregulated compared with the KK-A y group ( Figure 8B). Furthermore, as shown in Figure 9  Note: Variation ratio, the ratio of spot intensity of the KK-A y mice + dl-PHPB group to that of the same spot in the KK-A y group; " +"and " -" represent "upregulation" and "downregulation."

| PI3K/Akt/GSK-3β signaling pathway in the brain of KK-A y mice and KK-A y +dl-PHPB mice
Abbreviations: MW, molecular mass in kDa; pI, isoelectric point.
shown in Figure 10, PI3K, p-AKT (Ser473), p-GSK3β (Ser9) were all downregulated both in the cortex and hippocampus of KK-A y mice compared with C57 mice, but only p-GSK3β (Ser9) was significant.
After dl-PHPB treatment, all the three proteins were significantly upregulated compared with the KK-A y group. However, there were no differences between the expression of total Akt and GSK3β, and dl-PHPB also had no effect on the expression levels of the two proteins.

| DISCUSS ION
dl-PHPB is thought to be a promising candidate for the treatment of We used 2D-DIGE to conduct a proteomic study of brain tissues of KK-A y mice treated with and without dl-PHPB to identify proteins that might participate in the development of DE, as well as identify putative targets of dl-PHPB in the treatment of DE. More than 5000 protein spots were successfully detected in brain tissues of the two groups on each gel, and a total of 25 proteins showed significant changes (13 upregulated and 12 downregulated). These proteins were then categorized into five groups according to their functions based on the UniProt database ( Table 3). Most proteins were involved in energy metabolism, neuronal structure and trafficking, protein metabolism, stress and repair, and cell signaling.
Two of the differentially expressed proteins (GNAO1 and HINT1) were chosen to be validated via western blot. Furthermore, another four proteins (HAGH, EB1, DRP2, and LAMTOR2) that we confirmed to be altered in KK-A y mice in our previous study were also examined to observe their variation after dl-PHPB treatment, even though no changes were detected in the proteomic experiment. All six proteins were downregulated in brain tissues in the KK-A y group compared with control group mice, and dl-PHPB treatment could partially reverse the decrease in these six proteins.
Guanine nucleotide-binding protein G(o) subunit alpha (GNAO1), is a component of G protein transmembrane signaling. 33 It is highly enriched in the CNS and can inhibit neurotransmitter release and exhibit a protective effect on the CNS. Mutations in GNAO1 were previously associated with neurologic pathophysiology. 34 A previous proteomic analysis of AD also showed that GNAO1 was altered in the human hippocampus and inferior parietal lobe. 33,35 Furthermore, GNAO1 was also found to be downregulated in the retina of diabetic mice. 36 GNAO1 gene's mutations can cause a complex constellation of neurological disorders including epilepsy, movement disorders, and developmental delay. 37 In our study, proteomic and western blot analyses both showed that GNAO1 was downregulated in the brains of KK-Ay mice, and dl-PHPB could reverse this decrease in GNAO1. This result indicated that dl-PHPB might improve the spatial memory of KK-A y mice through regulation of transmembrane signaling and cellular processes.
Histidine triad nucleotide-binding protein 1 (HINT1) belongs to the histidine triad superfamily, which contains a highly conserved Note: Variation ratio, the ratio of spot intensity of the KK-A y mice + dl-PHPB group to that of the same spot in the KK-A y group; "+" and "-" represent "upregulation" and "downregulation." Abbreviations: MW, molecular mass in kDa; pI, isoelectric point.
His-X-His-X-His-XX motif (X indicates a hydrophobic amino acid). 38 HINT1 is widely expressed in different tissues, such as the liver, kidney, and brain. 39 The function of this protein is not fully known, but it is reported to be an inhibitor of Ca 2+ -dependent protein kinase C (PKC) and may play a role in tumor suppression. 40,41 The downregulation of HINT1 has also been reported in diabetes, AD, and schizophrenia. [42][43][44] All these results suggest that HINT1 may be of importance to neuronal function, the exact physiological and Hydroxyacylglutathione hydrolase (HAGH) belongs to the glyoxalase system and helps prevent the formation of AGEs. 45 This protein was reported to be altered in diabetic patients, animal models of diabetes, 45,46 and in AD patients. 47 DRP2, also known as collapsin response mediator protein 2 (CRMP2), is a neuron-specific phosphoprotein that plays roles in axon growth and elongation, establishing and maintaining neuronal polarity, and promoting microtubule assembly. 48 will not only lead to insulin deficiency and insulin resistance in DM patients, but also lead to tau hyperphosphorylation in the brain of AD patents. 58,59 Activation of PI3K/Akt pathway was effective against AD-like lesions 60 and could also prevent DM-related pathological changes in DM mice. 61 These findings are consistent with our experimental results. In KK-A y mice, the expressions of phosphor-Akt and PI3K were obviously lower than those in control C57 mice, but phosphorylated GSK-3β at Ser9 site was significantly higher. In contrast, dl-PHPB significantly activated the This study showed for the first time that dl-PHPB administration could significantly improve spatial learning and memory deficits in a F I G U R E 9 HINT1, HAGH, EB1, DRP2, and LAMTOR2 take part in PI3K/Akt/ GSK3β signaling pathway. HINT can suppress the activity of GSK3β and play a role in neuroprotection. LAMTOR2 is crucial for Akt/mTOR signaling activation and influences endosomal biogenesis and receptor trafficking. HAGH takes part in the scavenging system of methylglyoxal (MG) which is by PI3K/AKT/mTOR signaling. DRP2 is a brain-specific substrate of GSK3 and Cyclin-Dependent Kinase 5 (CDK 5) and takes part in axon elongation. GSK-3β regulates the activity of CLIP-associating protein 2 (CLASP2) and the interaction of CLASP2 and EB1 is necessary for the regulation of microtubule dynamics.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.